Project Summary/Abstract Allostery, in which a signal is transmitted from one part of a protein structure to another, plays critical roles in regulating many biological processes. Although allostery is traditionally associated with oligomers, it is now thought to be an inherent property of essentially all proteins. However, our fundamental understanding of allostery remains incomplete. This gap prevents us from testing mechanistic hypotheses about allosteric regulation of function in biomedically important proteins such as the dynamic Protein Tyrosine Phosphatase enzymes. The 100+ human PTPs play critical, diverse, and specific roles in signal transduction and diseases, with various individual PTPs linked to diabetes, cancers, or neurological disorders. However, unlike the complementary Protein Tyrosine Kinases, there are no approved drugs for the relatively understudied PTPs. My recent work broke new ground in understanding PTP regulation by revealing a surprisingly extensive allosteric network in the catalytic domain of the archetypal PTP family member, PTP1B, that transmits perturbations by small molecules or mutations from a contiguous regulatory interface to the dynamic active site to inhibit catalysis. Building off this result for one PTP, my group will explore how allosteric networks are ?re- wired? in a menagerie of different PTPs, which all have very similar catalytic domains that interact with highly varied regulatory domains. We will study a select group of 8-10 PTPs selected for therapeutic relevance, experimental tractability, and sequence diversity, including but not limited to TCPTP, CD45, STEP, LYP, and SHP2. To map the correlated conformational changes that underlie allostery in these proteins, we will develop new multidataset approaches in X-ray crystallography that use perturbation series (temperature, pressure, humidity, etc.) to elicit and model mechanical responses in the protein that underlie coupling between remote functional sites. To elucidate how these specific allosteric signatures arose during evolution despite the constraints of a catalytic domain architecture shared by all PTPs, we will create chimeras of catalytic and regulatory domains from different PTPs, and explore the extent to which each must be customized for the other using protein design calculations and selection experiments. The results will provide general insights into how modular proteins can be recombined to achieve orthogonal functions, not only in natural evolution but also for creating biosensors or computational molecular circuits in synthetic biology. This work will lead to hypotheses about unique allosteric weak points in individual PTPs, which we will test using new high-throughput, X-ray- based small-molecule screening methods and followup chemical biology experiments to generate new footholds for specific therapeutic development. Overall, the proposed work will have broad implications for fundamental questions in biophysics, including how conformational ensembles drive protein function, and will open new doors in allosteric therapeutic development and protein engineering.